Triple-Loop Dynamic DNA Nanonetwork with Cascaded Signal Amplification toward Self-Validating MicroRNA Biosensing

MicroRNAs, critical mediators of epigenetic regulation, govern vital biological processes with dysregulation linked to diseases, yet their low abundance and high sequence homology impose dual challenges on detection sensitivity and specificity. To overcome these limitations, a fluorescence and photoelectrochemical dual-mode biosensor was engineered, employing an entropy-driven circuit (EDC) and DNAzyme-based triple-loop cascade nanonetwork. Target recognition initiates EDC-mediated release of the resulting products, simultaneously activating DNAzyme I and DNAzyme II to produce target analogues (T*), thereby establishing a dual-amplification loop in which T* triggers EDC feedback to form a cascaded amplification nanonetwork. Signal transduction was enabled by CdS quantum dots (QDs), with the response synergistically amplified by g-C₃N₄ nanosheets to achieve enhanced photoelectrochemical performance. Furthermore, the elaborately designed positive feedback triple amplification system substantially improves the sensor's detection sensitivity. Specifically, the EDC amplification module maximizes the utilization of DNA strands, thereby significantly enhancing both operational efficiency and cost-effectiveness. Additionally, the precisely engineered triple amplification nanonetwork combined with magnetic extraction substantially improves the sensor's resistance to interference from potential coexisting substances, thereby enhancing its specificity and stability. Moreover, the dual-signal output mechanism in this design enables mutual validation of the detection results, further reinforcing the reliability and credibility of the assay outcomes.